leupeptins and Lung-Neoplasms

Arl4c is overexpressed in several cancer tissues and is involved in cancer development. Nevertheless, the exact mechanism that regulates Arl4c expression in lung cancer has not been fully elucidated. The aim of this study was to investigate the regulatory mechanism of Arl4c and to explore potential chemotherapeutic drugs targeting Arl4c.. Immunohistochemistry was used to examine Arl4c expression levels in human lung adenocarcinoma cancer specimens. Protein expression was detected by western blot. Overexpression of Arl4c-Flag protein was used to detect the ubiquitination of Arl4c. A short interfering RNA against Arl4c was used for gene silencing.. Arl4c was overexpressed in lung cancer tissues, and knockdown of Arl4c expression by siRNA decreased lung cancer A549 and 95-D cell proliferation. In addition, Arl4c expression was downregulated via inhibition of the AKT pathway in A549 and 95-D cells, whereas exposure to benzo (a) pyrene (a carcinogen in smoke) increased Arl4c expression in 16HBE cells via AKT activation. Finally, we found that chemotherapy drug hydroxycamptothecin (HCPT) could decrease Arl4c expression levels by inhibiting the activation of the AKT pathway in A549 and 95-D cells. Moreover, accumulation of ubiquitinated Arl4c protein was increased by HCPT and LY294002 (an AKT inhibitor) treatment whereas the proteasome inhibitor MG-132 attenuated the inhibitory effect of HCPT and LY294002 on Arl4c expression.. Here, we highlighted the AKT pathway as an important regulatory pathway for Arl4c expression in lung cancer cells and identified HCPT as a promising drug for lung adenocarcinoma treatment that functioned by targeting Arl4c expression.

Topics: A549 Cells; Adenocarcinoma; Adenocarcinoma of Lung; ADP-Ribosylation Factors; Blotting, Western; Camptothecin; Cell Line, Tumor; Chromones; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Leupeptins; Lung Neoplasms; Morpholines; Proto-Oncogene Proteins c-akt; Signal Transduction; Smoking

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer and accounts for 85% of all lung carcinomas. The hepatocyte growth factor receptor (c-Met) has been considered as a potential therapeutic target for NSCLC. Proteasome inhibition induces cell apoptosis and has been used as a novel therapeutic approach for treating diseases including NSCLC; however, the effects of different proteasome inhibitors on NSCLC have not been fully investigated. The aim of this study is to determine a precise strategy for treating NSCLC by targeting c-Met using different proteasome inhibitors. Three proteasome inhibitors, bortezomib, MG132, and ONX 0914, were used in this study. Bortezomib (50 nM) significantly reduced c-Met levels and cell viability in H1299 and H441 cells, while similar effects were observed in H460 and A549 cells when a higher concentration (100 nM) was used. Bortezomib decreased c-Met gene expression in H1299 and H441 cells, but it had no effect in A549 and H460 cells. MG-132 at a low concentration (0.5 M) diminished c-Met levels in H441 cells, while neither a low nor a high concentration (20 M) altered c-Met levels in A549 and H460 cells. A higher concentration of MG-132 (5 M) was required for decreasing c-Met levels in H1299 cells. Furthermore, MG-132 induced cell death in all four cell types. Among all the four cell lines, H441 cells expressed higher levels of c-Met and appeared to be the most susceptible to MG-132. MG-132 decreased c-Met mRNA levels in both H1299 and H441 cells. ONX 0914 reduced c-Met levels in H460, H1299, and H441 cells but not in A549 cells. c-Met levels were decreased the most in H441 cells treated with ONX 0914. ONX 0914 did not alter cell viability in H441; however, it did induce cell death among H460, A549, and H1299 cells. This study reveals that different proteasome inhibitors produce varied inhibitory effects in NSCLS cell lines.

Topics: Antineoplastic Agents; Apoptosis; Bortezomib; Carcinoma, Non-Small-Cell Lung; Cell Death; Cell Line, Tumor; Cell Proliferation; Cell Survival; Humans; Leupeptins; Lung Neoplasms; Oligopeptides; Proteasome Inhibitors; Proto-Oncogene Proteins c-met

Alterations in KEAP1/ NF-E2 p45-related factor 2 (NFE2L2/Nrf2) signaling pathway have been reported in 23% lung adenocarcinoma patients, suggesting that deregulation of the pathway is a major cancer driver. Here we report that mitogen-activated protein (MAP) kinase phosphatase 1 (MKP-1) drives tumor growth and drug resistance by up regulating transcription factor Nrf2. In non-small cell lung cancer (NSCLC) cells and xenografts, MKP-1 knockdown triggered the down-regulation of the metabolic enzymes and cytoprotective proteins, which are the target genes of Nrf2. Consequently, the cell growth was markedly inhibited with decrease of tumor metabolisms and GSH contents. Moreover, MKP-1 silencing sensitized NSCLC cells to cisplatin treatment. Mechanistically, MKP-1 inhibited the ubiquitylation of Nrf2 via a direct interaction with the transcription factor. Nrf2 was hence stabilized and its transcriptional program was activated. Notably, Nrf2 elevated MKP-1 expression at transcriptional level. In human lung adenoma tumor samples, high levels of expression of MKP-1, Nrf2, and its target gene heme oxygenase 1 were closely correlated. Thus, MKP-1 and Nrf2 form a forward feedback loop in lung cancer cells, which stabilizing and activating Nrf2 to promote anabolic metabolism and GSH biosynthesis. This study uncovers a novel role of MKP-1 in the malignant evolution of cancers.

Topics: Adenoma; Animals; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cell Survival; Drug Resistance, Neoplasm; Dual Specificity Phosphatase 1; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Leupeptins; Lung Neoplasms; Male; Mice; Mice, Nude; Neoplasms, Experimental; NF-E2-Related Factor 2

Natural killer (NK) cells recognize stress‑activated NK group 2, member D (NKG2D) ligands in tumors. In the present study, the expression levels of NKG2D ligands were examined in four lung cancer cell lines (A549, PLA801D, NCI‑H157 and NCI‑H520). In the A549 cells, the expression of MHC class I polypeptiderelated sequence (MIC)A/B and UL16 binding protein (ULBP)1 was weak, the expression of ULBP2 was typical, and neither ULBP3 nor ULBP4 were expressed. The mechanism underlying the regulatory effect of a cancer treatment agent on the expression of NKG2D ligands was investigated using the proteasome inhibitor MG132. Following treatment for 8 h with MG132, the transcription levels of MICB and ULBP1 were upregulated 10.62‑ and 11.09‑fold, respectively, and the expression levels of MICB and ULBP1 were increased by 68.18 and 23.65%, respectively. Notably, MICB exhibited significant time‑dependent change. MG132 increased the transcription of MICB by acting at a site in the 480‑bp MICB upstream promoter. The activity of the MICB promoter was upregulated 1.77‑fold following treatment with MG132. MG132 treatment improved the cytotoxicity of NK cells, which was partially blocked by an antibody targeting NKG2D, and more specifically the MICB molecule. The expression of MICB induced by MG132 was inhibited by KU‑55933 [ataxia telangiectasia mutated (ATM) kinase inhibitor], wortmannin (phosphoinositide 3 kinase inhibitor) and caffeine (ATM/ATM‑Rad3‑related inhibitor). The phosphorylation of checkpoint kinase 2 (Chk2), an event associated with DNA damage, was observed following treatment with MG132. These results indicated that MG132 selectively upregulates the expression of MICB in A549 cells, and increases the NKG2D‑mediated cytotoxicity of NK cells. The regulatory effect of MG132 may be associated with the activation of Chk2, an event associated with DNA damage. The combination of MG132 with NK cell immunotherapy may have a synergistic effect that improves the therapeutic effect of lung cancer treatment.

Topics: A549 Cells; Antineoplastic Agents; DNA Damage; Gene Expression Regulation, Neoplastic; Histocompatibility Antigens Class I; Humans; Leupeptins; Lung Neoplasms; Promoter Regions, Genetic; Transcriptional Activation

Our previous study suggested the anti-tumor activity of sepia ink oligopeptide (SIO). Here we sought to investigate the underlying molecular mechanism.. Cell proliferation was evaluated by cell counting kit-8 (CCK-8) assay. Cell apoptosis was determined by Annexin V/Propidium Iodide (PI) staining. The mitochondria pathway was characterized by quantification of Bcl-2, Bax, Caspase-9 and Cyto-C. The death receptor pathway was analyzed by determinement of Fas, Caspase-8 and NIK. The endoplasmic reticulum (ER)-dependent pathway was determined by measurement the expression of CHOP, Caspase-12, GRP78 and Calpain. The associated gene expression was quantified by RT-PCR and protein level was determined by immunoblotting.. We demonstrated treatment with structurally modified SIO (CSIO, 5 µM) significantly inhibited cell proliferation and induced apoptosis in lung cancer cell line A549. The mitochondrial pathway, death receptor pathway and ER stress induced apoptosis were stimulated upon CSIO treatment. The administration with respective inhibitors including midiv-1 (50 µM for 2 h), PDTC (20 µM PDTC for 30 min) and ALLN (20 mM ALLN for 5 h) readily reversed the apoptosis inducing effect of CSIO.. Our data demonstrates that CSIO is capable of induction apoptosis in lung cancer cell line, which is mediated by all three classical apoptotic pathways. Our results warrant further in vivo investigations of the anti-tumor potential of CSIO.

Topics: A549 Cells; Animals; Apoptosis; bcl-2-Associated X Protein; Calpain; Caspase 12; Caspase 8; Caspase 9; Cell Proliferation; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; fas Receptor; Humans; Ink; Leupeptins; Lung Neoplasms; Mitochondria; Oligopeptides; Proline; Proto-Oncogene Proteins c-bcl-2; Sepia; Thiocarbamates; Transcription Factor CHOP

EZH2 (the Enhancer of Zeste Homolog 2), as a key epigenetic regulator and EMT inducer, participates in a variety of cancer metastasis. EZH2 stability is regulated by several types of post-translational modifications (PTMs).The long non-coding RNAs (lncRNA) have been implicated to have critical roles in multiple carcinogenesis through a wide range of mechanisms, including modulating the stability of proteins. To date, whether the stability of EZH2 protein is regulated by lncRNAs remains unexplored. Here we report the discovery of ANCR modulating the stability of EZH2, and hence in the invasion and metastasis of breast cancer cells. We determined that ANCR potentiated the CDK1-EZH2 interaction, which then increased the intensity of phosphorylation at Thr-345 and Thr-487 sites of EZH2, facilitating EZH2 ubiquitination and hence its degradation. Moreover, we also uncover ANCR is an important player in breast cancer progression and metastasis mainly through decreasing EZH2 stability. More specifically, we initially found that ANCR level was lower in breast cancer tissues and breast cancer cell lines, in contrast to their normal counterparts. We then demonstrated that knockdown of ANCR induced an EMT program and promoted cell migration and invasion in MCF10A (epithelial cells), whereas ectopic expression of ANCR repressed breast cancer cells migration and invasion. Furthermore, we validated in a nude mouse model that overexpression of ANCR in highly malignant and invasive MDA-MB-231 breast cancer cells significantly reduced the ability of the cells to form tumors and prevented the lung metastasis in vivo. Based on these data, our findings define a new mechanism underlying modulation of EZH2 stability by linking ANCR interaction with EZH2 to promote its phosphorylation that facilitates EZH2 degradation and suppresses breast cancer progression.

Topics: Animals; Breast Neoplasms; Cadherins; CDC2 Protein Kinase; Cell Line, Tumor; Cell Movement; Cycloheximide; Enhancer of Zeste Homolog 2 Protein; Female; Gene Expression Regulation, Neoplastic; Humans; Leupeptins; Lung Neoplasms; MCF-7 Cells; Mice; Mice, Inbred BALB C; Mice, Nude; Proteasome Endopeptidase Complex; Protein Binding; RNA, Long Noncoding; Ubiquitin

Tumour metastasis, the spread of cancer cells from the original tumour site followed by growth of secondary tumours at distant organs, is the primary cause of cancer-related deaths and remains poorly understood. Here we demonstrate that inhibition of CDK4/6 blocks breast tumour metastasis in the triple-negative breast cancer model, without affecting tumour growth. Mechanistically, we identify a deubiquitinase, DUB3, as a target of CDK4/6; CDK4/6-mediated activation of DUB3 is essential to deubiquitinate and stabilize SNAIL1, a key factor promoting epithelial-mesenchymal transition and breast cancer metastasis. Overall, our study establishes the CDK4/6-DUB3 axis as an important regulatory mechanism of breast cancer metastasis and provides a rationale for potential therapeutic interventions in the treatment of breast cancer metastasis.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Movement; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Disease Models, Animal; Endopeptidases; Female; Gene Expression Regulation, Neoplastic; Humans; Leupeptins; Liver Neoplasms; Lung Neoplasms; MCF-7 Cells; Mice; Ovarian Neoplasms; Piperazines; Pyridines; RNA, Small Interfering; Signal Transduction; Snail Family Transcription Factors; Triple Negative Breast Neoplasms; Xenograft Model Antitumor Assays

The adenovirus vector-based cancer gene therapy is controversial. Low transduction efficacy is believed to be one of the main barriers for the decreased expression of coxsackie and adenovirus receptor (CAR) on tumor cells. However, the expression of CAR on primary tumor tissue and tumor tissue survived from treatment has still been not extensively studied. The present study analyzed the adenovirus infection rates and CAR expression in human lung adenocarcinoma cell line A549 and its cisplatin-resistant subline A549/DDP. The results showed that although the CAR expression in A549 and A549/DDP was not different, compared with the A549, A549/DDP appeared obviously to reject adenovirus infection. Moreover, we modified CAR expression in the two cell lines with proteasome inhibitor MG-132 and histone deacetylase inhibitor trichostatin A (TSA), and analyzed the adenovirus infection rates after modifying agent treatments. Both TSA and MG-132 pretreatments could increase the CAR expression in the two cell lines, but the drug pretreatments could only make A549 cells more susceptible to adenovirus infectivity.

Topics: A549 Cells; Adenocarcinoma; Adenocarcinoma of Lung; Adenoviridae; Adenoviridae Infections; Cell Line, Tumor; Cisplatin; Coxsackie and Adenovirus Receptor-Like Membrane Protein; Drug Resistance, Neoplasm; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Leupeptins; Lung Neoplasms; Proteasome Inhibitors

ING5 can interact with p53, thereby inhibiting cell growth and inducing apoptosis. We found that ING5 overexpression not only inhibited proliferation, migration, and invasion, but also induced G2 arrest, differentiation, autophagy, apoptosis, glycolysis and mitochondrial respiration in lung cancer cells. ING5 transfection up-regulated the expression of Cdc2, ATG13, ATG14, Beclin-1, LC-3B, AIF, cytochrome c, Akt1/2/3, ADFP, PFK-1 and PDPc, while down-regulated the expression of Bcl-2, XIAP, survivin,β-catenin and HXK1. ING5 transfection desensitized cells to the chemotherapy of MG132, paclitaxel, and SAHA, which paralleled with apoptotic alteration. ING5 overexpression suppressed the xenograft tumor growth by inhibiting proliferation and inducing apoptosis. ING5 expression level was significantly higher in normal tissue than that in lung cancer at both protein and mRNA levels. Nuclear ING5 expression was positively correlated with ki-67 expression and cytoplasmic ING5 expression. Cytoplasmic ING5 expression was positively associated with lymph node metastasis, and negatively with age, lymphatic invasion or CPP32 expression. ING5 expression was different in histological classification: squamous cell carcinoma > adenocarcinoma > large cell carcinoma > small cell carcinoma. Taken together, our data suggested that ING5 downregulation might involved in carcinogenesis, growth, and invasion of lung cancer and could be considered as a promising marker to gauge the aggressiveness of lung cancer. It might be employed as a potential target for gene therapy of lung cancer.

Topics: A549 Cells; Aged; Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Humans; Hydroxamic Acids; Kaplan-Meier Estimate; Leupeptins; Lung Neoplasms; Male; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Molecular Targeted Therapy; Paclitaxel; Transcription Factors; Tumor Suppressor Proteins; Vorinostat; Xenograft Model Antitumor Assays

Lung cancer remains the top cancer killer worldwide, with squamous cell carcinoma (SCC) as the second commonest histologic subtype. Arsenic trioxide (ATO) was previously shown to suppress growth of lung cancer. Fibroblast growth factor receptor (FGFR) amplification was recently demonstrated in lung SCC, with specific FGFR inhibitor (e.g. PD173074) developed as a potential targeted therapy. Therefore the combination effects of ATO and PD173074 in SCC was studied.. The combination of ATO/PD173074 was studied in a proof-of-principle model using a lung SCC cell line with FGFR1 overexpression: SK-MES-1. The effects of ATO and/or PD173074 on cell viability and protein expression were studied by MTT assay and Western blot respectively. Cell cycle analysis, phosphatidylserine externalization and mitochondrial membrane depolarization were monitored by flow cytometry. FGFR1 knockdown was performed with siRNAs. Proteasome inhibitor (MG-132) was used to study the degradation mechanism. In vivo effect of ATO and/or PD173074 was investigated using a nude mice xenograft model.. Combined ATO/PD173074 reduced cell viability along with increased sub-G1 population, phosphatidylserine externalization and mitochondrial membrane depolarization more significantly than single treatments. Downregulation of FGFR1, p-Akt, Akt, p-Src, Src, p-c-Raf, c-Raf, Erk and survivin as well as upregulation of p-Erk and cleaved PARP were observed upon ATO and/or PD treatment. MG-132 partially reversed the degradation of Akt, Src, c-Raf and Erk induced by ATO/PD, suggestive of ubiquitin-independent proteasome-dependent degradation. However, the mechanism of FGFR1 downregulation remained unknown. Downregulation of FGFR1, Akt, Src, c-Raf and Erk as well as cleaved PARP elevation induced by ATO and/or PD were confirmed in vivo.. Massive protein degradation (FGFR1, Akt, Src, c-Raf and Erk) was induced by ATO and/or PD173074 treatment mainly mediated by activation of proteasomal degradation in SCC cell line SK-MES-1 in vitro and in vivo.

Topics: Animals; Antineoplastic Agents; Apoptosis; Arsenic Trioxide; Arsenicals; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Survival; Cysteine Proteinase Inhibitors; Drug Therapy, Combination; Female; Leupeptins; Lung Neoplasms; Mice; Mice, Nude; Oxides; Pyrimidines; Receptor, Fibroblast Growth Factor, Type 1; Xenograft Model Antitumor Assays

14-3-3 proteins are a family of highly conserved polypeptides that interact with a large number of proteins and play a role in a wide variety of cellular processes. 14-3-3 proteins have been demonstrated overexpressed in several cancers and serving as potential oncogenes. In a previous study we showed one isoform of the 14-3-3 family, 14-3-3γ was negatively regulated by p53 through binding to its promoter and inhibiting its transcription. In the present study we investigated both p53 and 14-3-3γ protein levels in human lung cancerous tissues and normal lung tissues. We found 14-3-3γ expression correlated to p53 overexpression in lung cancer tissues. Ecotopic expression of wild-type p53, but not mutant p53 (R175H) suppressed both endogenous and exogenous 14-3-3γ in colon and lung cancer cell lines. Further examination demonstrated that p53 interacted with C-terminal domain of 14-3-3γ and induced 14-3-3γ ubiquitination. MG132, a specific inhibitor of the 26S proteasome, could block the effect of p53 on 14-3-3γ protein levels, suggesting that p53 suppressed 14-3-3γ by stimulating the process of proteasome-mediated degradation of 14-3-3γ. These results indicate that the inhibitory effect of p53 on 14-3-3γ is mediated also by a post-transcriptional mechanism. Loss of p53 function may result in upregulation of 14-3-3γ in lung cancers.

Topics: 14-3-3 Proteins; Apoptosis; Gene Expression Regulation, Neoplastic; Humans; Leupeptins; Lung; Lung Neoplasms; Mutation; Proteasome Endopeptidase Complex; Proteolysis; Tumor Suppressor Protein p53; Ubiquitination

Stabilization of Bcl-2 protein by paxillin (PXN)-mediated ERK activation was recently reported to cause an unfavorable response to 5-Fluorouracil-based chemotherapy. Here, we present evidence from cell and animal models to demonstrate that stabilization of Bcl-2 protein by phosphorylation at Serine 87 (pBcl-2-S87) via PXN-mediated ERK activation is responsible for cancer cell invasiveness and occurs via upregulation of MMP2 expression. Immunostainings of 190 tumors resected from colorectal cancer patients indicated that PXN expression was positively correlated with Bcl-2, pBcl-2-S87, and MMP2 expression. A positive correlation of pBcl-2-S87 with Bcl-2 and MMP2 was also observed in this study population. Patients with high PXN, Bcl-2, pBcl-2-S87, and MMP2 had poor overall survival (OS) and shorter relapse free survival (RFS). In conclusion, PXN promotes Bcl-2 phosphorylation at Serine 87 via PXN-mediated ERK activation, and its stabilization associated with increased tumor formation efficacy in mice and poor patient outcome in colorectal cancer patients.

Topics: Animals; Benzimidazoles; Bridged Bicyclo Compounds, Heterocyclic; Cell Line, Tumor; Colorectal Neoplasms; Dasatinib; Enzyme Activation; Enzyme Induction; Extracellular Signal-Regulated MAP Kinases; Heterografts; Humans; Kaplan-Meier Estimate; Leupeptins; Lung Neoplasms; Matrix Metalloproteinase 2; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasm Proteins; Paxillin; Phosphorylation; Proportional Hazards Models; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-bcl-2; Recombinant Fusion Proteins; RNA Interference; RNA, Small Interfering; Sulfonamides

Radiotherapy is a common treatment modality for lung cancer, however, radioresistance remains a fundamental barrier to attaining the maximal efficacy. Cancer cells take advantage of the ubiquitin-proteasome system (UPS) for increased proliferation and decreased apoptotic cell death. MG132 (carbobenzoxyl-leucinyl-leucinyl-leucinal‑H), a specific and selective reversible inhibitor of the 26S proteasome, has shown anticancer effect in multiple types of cancers. Previously, we have reported that MG132 enhances the anti‑growth and anti-metastatic effects of irradiation in lung cancer cells. However, whether MG132 can enhance the radiosensitivity in lung cancer cells in vitro and in vivo is still unknown. In this study, we found that MG132 increased apoptosis and dicentric chromosome ratio of A549 and H1299 cells treated by irradiation. Radiation-induced NF-κB expression and IκBα phosphorylation was attenuated in MG132 plus irradiation-treated cells. The in vivo model of H1299 xenografts of nude mice showed that the tumor size of MG132 plus irradiation treated xenografts was smaller than that of irradiation, MG132 or the control group. Moreover, MG132 plus irradiation group showed significant reduced Ki67 expression. Taken together, these results demonstrate that MG132 enhances the radiosensitivity through multiple mechanisms in vitro and in vivo.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Cell Survival; Gene Expression Regulation, Neoplastic; Humans; In Vitro Techniques; Ki-67 Antigen; Leupeptins; Lung Neoplasms; Mice; Radiation-Sensitizing Agents; Tumor Burden; Xenograft Model Antitumor Assays

The current treatment for advanced nonsmall cell lung cancer (NSCLC) remains unsatisfactory due to resistance to chemotherapy and ionizing radiation. The ubiquitin-proteasome system (UPS) regulates multiple cellular processes that are crucial for the proliferation and survival of all kinds of cells. Carbobenzoxyl-leucinyl-leucinyl-leucinal-H (MG132), a specific and selective reversible inhibitor of the 26S proteasome, represents a novel approach for cancer therapy. However, whether MG132 can potentiate the effect of radiation against the growth and metastasis of NSCLC is not clear. We found that MG132 inhibited the proliferation of human NSCLC cell lines (A549 and H1299) in a dose- and time-dependent manner by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Then MG132 at a nontoxic dose (100 nM) was selected for following studies. Pretreatment of A549 and H1299 cells with 100 nM MG132 before ionizing radiation (IR) potentiated the anticancer effect of IR. Moreover, pretreatment with 100 nM MG132 before IR-enhanced radiation induced cell cycle arrest by decreased CyclinD1 but increased Wee1 expression in A549 and H1299 cells. In addition, pretreatment of MG132 combined with IR significantly suppressed cell migration and invasion abilities in NSCLC cell lines, which was accompanied by decreased expression of matrix metalloproteinase (MMP)-2 and MMP-9 in NSCLC cell lines. Taken together, our results demonstrate that MG132 enhances the antigrowth and antimetastatic effects of irradiation in NSCLC cells by modulating expression of cell cycle and invasion- related genes.

Topics: Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Humans; Leupeptins; Lung Neoplasms; Neoplasm Metastasis; NF-kappa B; Nuclear Proteins; Proteasome Inhibitors; Protein-Tyrosine Kinases; Radiation Tolerance

High expression levels of cyclooxygenase-2 (COX-2) contribute a strong proliferative ability to human lung cancer cells, and this function is link to the epidermal growth factor receptor (EGFR) pathway, which was mediated by extracellular-signal-regulated kinase (ERK) and nuclear factor kappa B (NFκB). In this study, scutellarein, a flavonoid compound, was screened for proliferation inhibition at different concentrations (0, 5, 25 and 50 μM) at 24 h or 48 h in human lung cancer cell line A549. Results showed that A549 cell proliferation was inhibited by 50 μM scutellarein treatment in 24 h and 48 h of treatment. The expression levels of phosphorylated EGFR, phosphorylated ERK, phosphorylated NFκB and COX-2 were reduced in a dose-dependent manner after 24 h scutellarein treatments at different concentrations. Further, ERK inhibitor U0126 and NFκB inhibitor MG132 also inhibited A549 cell proliferation similar to 50 κM scutellarein treatment from 24 h to 48 h. The experimental results showed that scutellarein could inhibit proliferation of the human lung cancer cell line A549 through ERK and NFκB mediated by the EGFR pathway.

Topics: Antineoplastic Agents; Apigenin; Butadienes; Cell Line, Tumor; Cell Proliferation; Enzyme Inhibitors; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Humans; Leupeptins; Lung Neoplasms; MAP Kinase Signaling System; NF-kappa B; Nitriles

Mcl-1 and Bcl-xL, key anti-apoptotic proteins of the Bcl-2 family, have attracted attention as important molecules in the cell survival and drug resistance. In this study, we investigated whether inhibition of Bcl-xL influences cell growth and apoptosis against simultaneous treatment of resveratrol and clofarabine in the human malignant mesothelioma H-2452 cells. Resveratrol and clofarabine decreased Mcl-1 protein levels but had little effect on Bcl-xL levels. In the presence of two compounds, any detectable change in the Mcl-1 mRNA levels was not observed in RT-PCR analysis, whereas pretreatment with the proteasome inhibitor MG132 led to its accumulation to levels far above basal levels. The knockdown of Bcl-xL inhibited cell proliferation with cell accumulation at G2/M phase and the appearance of sub-G0/G1 peak in DNA flow cytometric assay. The suppression of cell growth was accompanied by an increase in the caspase-3/7 activity with the resultant cleavages of procaspase-3 and its substrate poly (ADP-ribose) polymerase, and increased percentage of apoptotic propensities in annexin V binding assay. Collectively, our data represent that the efficacy of resveratrol and clofarabine for apoptosis induction was substantially enhanced by Bcl-xL-lowering strategy in which the simultaneous targeting of Mcl-1 and Bcl-xL could be a more effective strategy for treating malignant mesothelioma.

Topics: Adenine Nucleotides; Antimetabolites, Antineoplastic; Apoptosis; Arabinonucleosides; bcl-X Protein; Caspase 3; Caspase 7; Cell Line, Tumor; Cell Proliferation; Clofarabine; G2 Phase Cell Cycle Checkpoints; Gene Knockdown Techniques; Humans; Leupeptins; Lung Neoplasms; M Phase Cell Cycle Checkpoints; Mesothelioma; Mesothelioma, Malignant; Myeloid Cell Leukemia Sequence 1 Protein; Resveratrol; RNA Interference; RNA, Messenger; RNA, Small Interfering; Stilbenes

Cellular senescence contributes to aging and decline in tissue function. p53 isoform switching regulates replicative senescence in cultured fibroblasts and is associated with tumor progression. Here, we found that the endogenous p53 isoforms Δ133p53 and p53β are physiological regulators of proliferation and senescence in human T lymphocytes in vivo. Peripheral blood CD8+ T lymphocytes collected from healthy donors displayed an age-dependent accumulation of senescent cells (CD28-CD57+) with decreased Δ133p53 and increased p53β expression. Human lung tumor-associated CD8+ T lymphocytes also harbored senescent cells. Cultured CD8+ blood T lymphocytes underwent replicative senescence that was associated with loss of CD28 and Δ133p53 protein. In poorly proliferative, Δ133p53-low CD8+CD28- cells, reconstituted expression of either Δ133p53 or CD28 upregulated endogenous expression of each other, which restored cell proliferation, extended replicative lifespan and rescued senescence phenotypes. Conversely, Δ133p53 knockdown or p53β overexpression in CD8+CD28+ cells inhibited cell proliferation and induced senescence. This study establishes a role for Δ133p53 and p53β in regulation of cellular proliferation and senescence in vivo. Furthermore, Δ133p53-induced restoration of cellular replicative potential may lead to a new therapeutic paradigm for treating immunosenescence disorders, including those associated with aging, cancer, autoimmune diseases, and HIV infection.

Topics: Adult; Aged; Autophagy; CD8-Positive T-Lymphocytes; Cell Division; Cells, Cultured; Cellular Senescence; Female; Gene Expression Regulation, Neoplastic; Humans; Leupeptins; Lung Neoplasms; Lymphocytes, Tumor-Infiltrating; Macrolides; Male; Middle Aged; Neoplasm Proteins; Protein Isoforms; Proteolysis; Recombinant Proteins; Transduction, Genetic; Tumor Microenvironment; Tumor Suppressor Protein p53; Young Adult

In this study, the effect of MG132 (carbobenzoxyl-leucinyl-leucinyl-leucinal-H) at a low dose on radiotherapeutic efficacy and its accurate mechanism of radiosensitization were investigated in human non-small cell lung cancer. The effect of MG132 on ionizing radiation (IR)-induced cytotoxicity, cell proliferation and survival of A549 cells was evaluated. The protein expression modulated by MG132 and IR were inspected by Western blot analysis. To determine in vivo radiotherapeutic efficacy, tumor growth delay was analyzed in a A549 tumor-bearing xenograft mouse model after single or repeated treatment of MG132 and/or IR. Induction of apoptosis and change of c-Myc expression in the tumor tissue was explored by histological analysis. MG132 at a non-toxic dose enhanced the radiation-induced cytotoxicity of A549 cells, accompanying a significant decrease of c-Myc expression. Suppression of c-Myc expression by small interfering RNA (siRNA) displayed enhancement of radiosensitivity similarly to MG132 treatment. Tumor growth in the xenograft mice was markedly delayed by systemic administration of MG132 combined with IR. In vivo down-regulation of c-Myc and increased induction of apoptosis were simultaneously observed in the tumor tissues followed by combinational treatment of MG132 and IR. The results reveal a novel mechanism for proteasome inhibitor-mediated radiosensitization in which c-Myc down-regulation is involved.

Topics: Animals; Carcinoma, Non-Small-Cell Lung; Cell Survival; Down-Regulation; Humans; Leupeptins; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Proteasome Inhibitors; Proto-Oncogene Proteins c-myc; Radiation-Sensitizing Agents; Xenograft Model Antitumor Assays

Cancer side population (SP) cells, which are often referred to as cancer stem cells, are thought to be responsible for lung cancer chemotherapy resistance, and currently no drug can specifically target these cells. We hypothesize low-molecular-weight heparin (LMWH) may affect the biological properties of SP cells and could be used to clinically target these cells. To test this, SP cells were isolated from cisplatin (DDP)-resistant lung adenocarcinoma A549/DDP cells by flow cytometric sorting. Compared to non-SP cells, SP cells formed increased numbers of colonies in vitro, and had a 1000-fold increase in tumorigenicity in vivo. Proliferation and apoptosis assays demonstrated LMWH had no significant effect on lung SP cell proliferation or apoptosis. However, LMWH reduced lung SP cell colony formation ability and protein expression of the multidrug transporter, ABCG2, by FACS and western blot analyses without affecting its mRNA levels by RT-PCR. Consistently, immunohistochemistry stainings of ABCG2 in LMWH-treated tumor tissues were significantly reduced compared with those in controls. Further, we found proteasomal inhibitor MG132, but not lysosomal inhibitors leupeptin and pepstatin A, could restore ABCG2 protein levels in LMWH-treated SP cells. These suggest LMWH ablates lung SP cell chemoresistance by proteasome-mediated reduction of ABCG2 protein levels without affecting its mRNA levels. We also determined LMWH combined with cisplatin could overcome cisplatin-resistance and induced lung SP cells apoptosis both in vitro and in vivo. This study provides an experimental basis for using a combination of LMWH, which targets lung SP cells, with chemotherapy to improve lung cancer survival.

Topics: Animals; Antineoplastic Agents; Apoptosis; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Biomarkers, Tumor; Cell Line, Tumor; Cell Proliferation; Cisplatin; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Heparin, Low-Molecular-Weight; Humans; Leupeptins; Lung Neoplasms; Male; Mice; Neoplasm Proteins; Neoplastic Stem Cells; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Proteolysis; Survival Analysis; Xenograft Model Antitumor Assays

Carbobenzoxy-Leu-Leu-leucinal (MG132) as a proteasome inhibitor has been shown to induce apoptotic cell death through formation of reactive oxygen species (ROS). In the present study, we evaluated the effects of MG132 on the growth of A549 lung cancer cells in relation to cell growth, ROS and glutathione (GSH) levels. Treatment with MG132 inhibited the growth of A549 cells with an IC(50) of approximately 20 microM at 24 hours. DNA flow cytometric analysis indicated that 0.5 approximately 30 microM MG132 induced a G1 phase arrest of the cell cycle in A549 cells. Treatment with 10 or 30 microM MG132 also induced apoptosis, as evidenced by sub-G1 cells and annexin V staining cells. This was accompanied by the loss of mitochondrial membrane potential (MMP; Delta psi m). The intracellular ROS levels including O(2) (*-) were strongly increased in 10 or 30 microM MG132-treated A549 cells but were down-regulated in 0.1, 0.5 or 1 microM MG132-treated cells. Furthermore, 10 or 30 microM MG132 increased mitochondrial O(2) (*- ) level but 0.1, 0.5 or 1 microM MG132 decreased that. In addition, 10 or 30 microM MG132 induced GSH depletion in A549 cells. In conclusion, MG132 inhibited the growth of human A549 cells via inducing the cell cycle arrest as well as triggering apoptosis, which was in part correlated with the changes of ROS and GSH levels. Our present data provide important information on the anti-growth mechanisms of MG132 in A549 lung cancer cells in relation to ROS and GSH.

Topics: Adenocarcinoma; Antineoplastic Agents; Apoptosis; Cell Cycle; Cell Proliferation; Enzyme Inhibitors; Humans; Leupeptins; Lung Neoplasms; Proteasome Endopeptidase Complex; Reactive Oxygen Species; Statistics, Nonparametric; Tumor Cells, Cultured

The inhibition of proteasome function has emerged as a useful strategy to maneuver apoptosis. In the present study, we evaluated the effects of MG132 as a proteasome inhibitor on the growth of Calu-6 lung cancer cells in relation to the cell cycle, cell death, reactive oxygen species (ROS) and glutathione (GSH) levels. MG132 dose-dependently inhibited the growth of Calu-6 cells at 24h. DNA flow cytometric analysis indicated that 1-30 microM MG132 induced an S phase arrest in Calu-6 cells. MG132 also induced apoptosis, which was accompanied by the loss of mitochondrial membrane potential (MMP; Deltapsi(m)). The pan-caspase inhibitor (Z-VAD) significantly rescued Calu-6 cells from MG132-induced cell death. The intracellular ROS levels including O(2)(-) were increased in MG132-treated Calu-6 cells. MG132 also increased GSH-depleted cell numbers in Calu-6 cells. Z-VAD significantly decreased O(2)(-) levels and GSH-depleted cell numbers in MG132-treated Calu-6 cells. In conclusion, MG132 inhibited the growth of Calu-6 cells via apoptosis and GSH depletion.

Topics: Antineoplastic Agents; Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Glutathione; Humans; Leupeptins; Lung Neoplasms; Membrane Potentials; Proteasome Inhibitors; Reactive Oxygen Species

MG132 as a proteasome inhibitor has been shown to induce apoptotic cell death through formation of reactive oxygen species (ROS). Here, we investigated the effects of N-acetyl cysteine (NAC; a well-known antioxidant), L-buthionine sulfoximine (BSO; an inhibitor of GSH synthesis) or diethyldithiocarbamate (DDC; an inhibitor of Cu/Zn-SOD) on MG132-treated Calu-6 or A549 lung cancer cells in relation to cell growth, ROS and GSH levels. MG132 inhibited the growth of Calu-6 and A549 cells at 24 h. MG132 induced apoptosis in both cell lines, which was accompanied by the loss of mitochondrial membrane potential (MMP; DeltaPsim). ROS levels including O(2)(.-) were increased in both MG132-treated lung cells. MG132 also induced GSH depletion in both lung cell types. Treatment with 10 microM BSO or 1 microM DDC affected ROS and GSH levels in MG132-treated Calu-6 cells. However, these changes did not influence cell growth and death in the cells. NAC prevented cell growth inhibition and death in MG132-treated lung cells, which was accompanied by decreased ROS, but not by decreased GSH depletion. In conclusion, the changes of ROS and GSH by MG132, NAC, BSO or DDC were partially related to cell growth and death in the lung cancer cell lines Calu-6 and A549.

Topics: Acetylcysteine; Apoptosis; Buthionine Sulfoximine; Cell Line, Tumor; Cell Proliferation; Ditiocarb; Glutathione; Humans; Leupeptins; Lung Neoplasms; Membrane Potential, Mitochondrial; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Reactive Oxygen Species

MG132, as a proteasome inhibitor, can induce apoptotic cell death through formation of reactive oxygen species (ROS). In this study, we investigated the effects of MAPK (MEK, JNK, and p38) inhibitors on MG132-treated A549 lung cancer cells in relation to cell growth, cell death, ROS, and glutathione (GSH) levels. Treatment with 10 microM MG132 inhibited the growth of A549 cells at 24 h. MG132 also induced apoptosis, which was accompanied by the loss of mitochondrial membrane potential (MMP; deltapsi(m)). ROS were not increased in MG132-treated A549 cells. MG132 increased GSH-depleted cell numbers and decreased GSH levels. MEK and JNK inhibitors did not strongly affect cell growth, cell death, ROS, and GSH levels in MG132-treated A549 cells. In contrast, p38 inhibitor reduced cell growth inhibition, apoptosis, and MMP (deltapsi(m)) loss by MG132. However, p38 inhibitor did not change ROS level and GSH content. In conclusion, MG132 inhibited the growth of A549 cells via apoptosis without formation of ROS. Treatment with p38 inhibitor rescued some cells from MG132-induced apotposis, which was not affected by ROS and GSH level changes.

Topics: Adenocarcinoma; Antineoplastic Agents; Apoptosis; Blotting, Western; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Enzyme Inhibitors; Glutathione; Humans; JNK Mitogen-Activated Protein Kinases; Leupeptins; Lung Neoplasms; Membrane Potential, Mitochondrial; p38 Mitogen-Activated Protein Kinases; Proteasome Inhibitors; Reactive Oxygen Species

MG132 (carbobenzoxy-Leu-Leu-leucinal) as a proteasome inhibitor has been shown to induce apoptotic cell death through formation of reactive oxygen species (ROS). In this study, we investigated the effects of MEK (mitogen-activated protein [MAP] kinase or extracellular signal-regulated kinase [ERK] kinase) or p38 inhibitor on MG132-treated Calu-6 lung cancer cells in relation to cell growth, cell death, ROS, and glutathione (GSH) levels. Treatment with 10 mumol/L MG132 inhibited the growth of Calu-6 cells at 24 hours. MG132 induced apoptosis in Calu-6 cells, which was accompanied by the loss of mitochondrial membrane potential (MMP; DeltaPsi(m)). ROS were increased in MG132-treated Calu-6 cells. MG132 also induced GSH depletion in Calu-6 cells. Treatment with MEK inhibitor did not significantly affect cell growth, cell death, ROS, and GSH levels in MG132-treated Calu-6 cells. Furthermore, MG132 increased the phosphorylation of p38 in Calu-6 cells at 1 and 24 hours. Treatment with p38 inhibitor significantly prevented cell growth inhibition, MMP (DeltaPsi(m)) loss and apoptosis in MG132-treated Calu-6 cells. This inhibitor increased ROS level and decreased GSH depletion in these cells. In conclusion, p38 inhibitor partially prevented Calu-6 cell death by MG132, which might be affected by GSH level changes.

Topics: Adenocarcinoma; Apoptosis; Blotting, Western; Calcium-Calmodulin-Dependent Protein Kinases; Cell Cycle; Cell Proliferation; Cysteine Proteinase Inhibitors; Flavonoids; Glutathione; Humans; Imidazoles; Leupeptins; Lung Neoplasms; Membrane Potential, Mitochondrial; p38 Mitogen-Activated Protein Kinases; Pyridines; Reactive Oxygen Species; Tumor Cells, Cultured

Proteasome inhibitors (PIs) are promising new therapeutic agents for treating non-small cell lung carcinoma (NSCLC). To investigate the mechanisms of action of PIs, we analyzed the proapoptotic activities of PIs (MG132 or Bortezomib) in NSCLC cells. We found that both MG132 (>1 microM) and Bortezomib (>0.025 microM) induced a significant apoptosis in NCI-H1703, a PI-sensitive NSCLC cell line, through initially activating the intrinsic apoptosis pathway, leading to the activation of a positive feedback mechanism (PFM), which then conveyed apoptosis signaling from the intrinsic pathway to the extrinsic pathway with formation of a signaling loop for maximal caspase activation. Mcl-1 and Noxa were identified to be the major anti-apoptotic and proapoptotic proteins, respectively, in PI-induced apoptosis and mutually exclusive in protein stability. Although the Mcl-1 protein was upregulated by proteasome inhibition, it was also subjected to caspase 3-dependent cleavage governed by the PFM. Moreover, it was revealed that Mcl-1 protein cleavage contributed to PFM-governed apoptosis in following inter-related ways: reducing the anti-apoptotic Mcl-1; generating the truncated proapoptotic Mcl-1(S); and inducing a shift of balance between Mcl-1 and Noxa. It was further manifested that tumor necrosis factor-related apoptosis-inducing ligand boosted MG132's proapoptotic activity through strengthening the PFM in both NCI-H1703 and NCI-H358, a PI-resistant NSCLC cell line. Therefore, this study provides a basis for enhancing the efficacy of PIs in treating NSCLC.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Non-Small-Cell Lung; Caspases; Cell Line, Tumor; Cysteine Proteinase Inhibitors; Feedback, Physiological; Humans; Leupeptins; Lung Neoplasms; Myeloid Cell Leukemia Sequence 1 Protein; Proteasome Inhibitors; Proto-Oncogene Proteins c-bcl-2; TNF-Related Apoptosis-Inducing Ligand

The tumor suppressor p53 is activated in response to many forms of cellular stress leading to cell cycle arrest, senescence or apoptosis. Appropriate sub-cellular localization is essential for modulating p53 function. We recently showed that p53 localizes to the nucleolus after proteasome inhibition with MG132 and this localization requires sequences within its carboxyl terminus. In the present study, we found that after treatment with MG132, p53 associates with a discrete sub-nucleolar component, the fibrillar center (FC), a region mainly enriched with RNA polymerase I. Moreover, we now demonstrate that this localization is an energy-dependent process as reduction of ATP levels prevents nucleolar localization. In addition, p53 sub-nucleolar accumulation is abolished when cells are subjected to various types of genotoxic stress. Furthermore, we show that monoubiquitination of p53, which causes it to localize to the cytoplasm and nucleoplasm, does not prevent the association of p53 with the nucleolus after MG132 treatment. Importantly, we demonstrate that p53 nucleolar association occurs in lung and bladder carcinomas.

Topics: Amino Acid Sequence; Carcinoma; Cell Nucleus; Cysteine Proteinase Inhibitors; Cytoplasm; Humans; Leupeptins; Lung Neoplasms; Molecular Sequence Data; Mutant Proteins; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Proto-Oncogene Proteins c-mdm2; Tumor Suppressor Protein p53; Ubiquitin; Ubiquitination; Urinary Bladder Neoplasms

We previously established a highly metastatic subline, LNM35, from the NCI-H460 lung cancer cell line, and demonstrated upregulation of a novel gene, CLCP1 (CUB, LCCL-homology, coagulation factor V/VIII homology domains protein), in LNM35 and lung cancer specimens. In this study, we focused on the potential roles of that gene in cancer metastasis. First, we established stable LNM35 RNAi clones, in which CLCP1 expression was suppressed by RNAi, and found that their motility was significantly reduced, although growth rates were not changed. Next, in vitro selection of a phage display library demonstrated that a phage clone displaying a peptide similar to a sequence within the Sema domain of semaphorin 4B (SEMA4B) interacted with LNM35. Immunoprecipitation experiments confirmed interaction of CLCP1 with SEMA4B, regulation of CLCP1 protein by ubiquitination and proteasome degradation enhanced in the presence of SEMA4B. These results are the first to indicate that CLCP1 plays a role in cell motility, whereas they also showed that at least one of its ligands is SEMA4B and that their interaction mediates proteasome degradation by CLCP1. Although the physiological role of the interaction between CLCP1 and SEMA4B remains to be investigated, this novel gene may become a target of therapy to inhibit metastasis of lung cancers.

Topics: Amino Acid Sequence; Cell Line; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cysteine Proteinase Inhibitors; Humans; Immunoblotting; Immunoprecipitation; Leupeptins; Lung Neoplasms; Membrane Proteins; Oligopeptides; Peptide Library; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Binding; RNA Interference; Semaphorins; Transfection; Tunicamycin; Ubiquitin

The PML tumor suppressor controls key pathways for growth suppression, induction of apoptosis, and cellular senescence. PML loss occurs frequently in human tumors through unknown posttranslational mechanisms. Casein kinase 2 (CK2) is oncogenic and frequently upregulated in human tumors. Here we show that CK2 regulates PML protein levels by promoting its ubiquitin-mediated degradation dependent on direct phosphorylation at Ser517. Consequently, PML mutants that are resistant to CK2 phosphorylation display increased tumor-suppressive functions. In a faithful mouse model of lung cancer, we demonstrate that Pml inactivation leads to increased tumorigenesis. Furthermore, CK2 pharmacological inhibition enhances the PML tumor-suppressive property in vivo. Importantly, we found an inverse correlation between CK2 kinase activity and PML protein levels in human lung cancer-derived cell lines and primary specimens. These data identify a key posttranslational mechanism that controls PML protein levels and provide therapeutic means toward PML restoration through CK2 inhibition.

Topics: Amino Acid Sequence; Amino Acid Substitution; Animals; Apoptosis; Carcinoma, Non-Small-Cell Lung; Casein Kinase II; Cell Line; Cell Line, Transformed; Cell Line, Tumor; Enzyme Activation; Enzyme Inhibitors; Genes, Tumor Suppressor; Green Fluorescent Proteins; Hemagglutinins; Humans; Leupeptins; Lung Neoplasms; Mice; Mice, Transgenic; Molecular Sequence Data; Neoplasm Proteins; NIH 3T3 Cells; Nuclear Proteins; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Promyelocytic Leukemia Protein; Proteasome Endopeptidase Complex; Protein Structure, Tertiary; Protein Subunits; RNA, Small Interfering; Sequence Deletion; Serine; Sorbitol; Transcription Factors; Transcriptional Activation; Triazoles; Tumor Suppressor Proteins; Ubiquitin

The cyclooxygenase-2 (COX-2) inhibitor celecoxib is an approved drug in the clinic for colon cancer chemoprevention and has been tested for its chemopreventive and therapeutic efficacy in various clinical trials. Celecoxib induces apoptosis in a variety of human cancer cells including lung cancer cells. Our previous work has shown that celecoxib induces death receptor 5 expression, resulting in induction of apoptosis and enhancement of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in human lung cancer cells. In the current study, we further show that celecoxib down-regulated the expression of cellular FLICE-inhibitory protein (c-FLIP), a major negative regulator of the death receptor-mediated extrinsic apoptotic pathway, through a ubiquitin/proteasome-dependent mechanism independent of COX-2 in human lung cancer cells. Overexpression of c-FLIP, particularly FLIP(L), inhibited not only celecoxib-induced apoptosis but also apoptosis induced by the combination of celecoxib and TRAIL. These results thus indicate that c-FLIP down-regulation also contributes to celecoxib-induced apoptosis and enhancement of TRAIL-induced apoptosis, which complements our previous finding that the extrinsic apoptotic pathway plays a critical role in celecoxib-induced apoptosis in human lung cancer cells. Collectively, we conclude that celecoxib induces apoptosis in human lung cancer cells through activation of the extrinsic apoptotic pathway, primarily by induction of death receptor 5 and down-regulation of c-FLIP.

Topics: Apoptosis; Blotting, Western; Carcinoma, Non-Small-Cell Lung; CASP8 and FADD-Like Apoptosis Regulating Protein; Celecoxib; Cell Line, Tumor; Cyclooxygenase 2; Cyclooxygenase Inhibitors; Down-Regulation; Drug Synergism; Flow Cytometry; Humans; Leupeptins; Lung Neoplasms; Pyrazoles; RNA, Small Interfering; Sulfonamides; TNF-Related Apoptosis-Inducing Ligand; Transfection; Ubiquitin

Non-small cell lung cancer frequently presents as a locally advanced disease. In this setting, radiation has a prominent role in cancer therapy. However, tumor adaptation to oxidative stress may lessen the efficacy of radiation therapy. Recent studies demonstrate that proteasome inhibitors increase the efficacy of radiation against a range of tumors. Although proteasome inhibition impacts on NF-kappaB translocation, the precise mechanism through which proteasome inhibitors induce tumor cell death and promote radiation efficacy remains unclear. The purpose of this study is to evaluate the potential of the proteasome inhibitor, MG-132, to improve the efficacy of radiation therapy and to determine whether its effect is linked to the suppression of the antioxidant enzyme, manganese superoxide dismutase (MnSOD). Human NSCLC (A549) cells were utilized both in vivo and in vitro to evaluate proteasome inhibition on radiation response. In vivo, mice that received combined treatments of 2.5 microg/g body weight MG-132 and 30 Gy demonstrated a delay in tumor regrowth in comparison to the 30 Gy control group. In vitro, clonegenic survival assays confirmed a dose-dependent enhancement of radiation sensitivity in combination with MG-132 and a significant interaction between the two. The levels of IkappaB-alpha, a NF-kappaB target gene and also an inhibitor of NF-kappaB nuclear translocation, decreased in a time-dependent manner following administration of MG-132 confirming the inhibition of the 26S proteasome. The MnSOD protein level was increased consistent with lower levels of IkappaB-alpha, confirming a NF-kappaB-mediated effect. Cells treated with radiation demonstrated an induction of MnSOD; however, the administration of MG-132 suppressed this induction These results support the hypothesis that proteasome inhibitors such as MG-132 can increase the efficacy of radiation therapy, in part, by suppression of cytoprotective NF-kappaB-mediated MnSOD expression.

Topics: Active Transport, Cell Nucleus; Animals; Antioxidants; Blotting, Western; Body Weight; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Combined Modality Therapy; Dose Fractionation, Radiation; Dose-Response Relationship, Drug; Female; Humans; I-kappa B Proteins; Leupeptins; Lung Neoplasms; Mice; Mice, Nude; Neoplasm Transplantation; Neoplasms; NF-kappa B; NF-KappaB Inhibitor alpha; Oxidative Stress; Protease Inhibitors; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Transport; Radiation Tolerance; Reactive Oxygen Species; Superoxide Dismutase; Time Factors

The p53 oncosuppressor protein is subject to negative regulation by MDM2, which efficiently inhibits its activity through an autoregulatory loop. In response to stress, however, p53 undergoes post-translational modifications that allow the protein to escape MDM2 control, accumulate, and become active. Recent studies have shown that, following DNA damage, the HIPK2 serine/threonine kinase binds and phosphorylates p53, inducing p53 transcriptional activity and apoptotic function. Here, we investigated the role of HIPK2 in the activation of p53 in the presence of MDM2. We found that HIPK2 rescues p53 transcriptional activity overcoming MDM2 inhibition, and that restoration of this p53 function induces apoptosis. Recovery of p53-dependent apoptosis is achieved by preventing p53 nuclear export and ubiquitination mediated by MDM2 in vitro and in vivo following genotoxic stress. These results shed new light on the mechanisms by which the HIPK2/p53 pathway promotes apoptosis and suppression of tumorigenesis.

Topics: Adenocarcinoma; Antineoplastic Agents; Apoptosis; Blotting, Western; Carrier Proteins; Cell Line, Tumor; Cell Nucleus; Cisplatin; Colonic Neoplasms; Cysteine Proteinase Inhibitors; DNA Damage; Humans; Leupeptins; Luciferases; Lung Neoplasms; Nuclear Proteins; Osteosarcoma; Precipitin Tests; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; Transcriptional Activation; Tumor Suppressor Protein p53; Ubiquitin

The role of the ubiquitin-proteasome pathway during roscovitine induced apoptosis was evaluated in the non-small cell lung carcinoma cell line MR65. To this end specific inhibitors of proteasome activity, MG132 and lactacystin were used. Addition of MG132 or lactacystin, 1 h prior to the addition of the CDK-inhibitor roscovitine to the cell cultures inhibited apoptosis significantly, as measured by PS exposure, cytokeratin 18 cleavage and caspase-3 activation. Furthermore, we show that inhibition of proteasome activation prior to induction of apoptosis by roscovitine prevents loss of mitochondrial inner transmembrane potential (DeltaPsim). In addition we found that MG132 and lactacystin prevent release of cytochrome c from the mitochondrion. In contrast to the above findings we see no effect of proteasome inhibition in Fas-mediated apoptosis. Taken together our data suggest a specific role for proteasomes very early in roscovitine-induced apoptosis, upstream from the caspase cascade and mitochondrion.

Topics: Acetylcysteine; Antineoplastic Agents; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cysteine Proteinase Inhibitors; Humans; Leupeptins; Lung Neoplasms; Membrane Potentials; Mitochondria; Proteasome Endopeptidase Complex; Purines; Roscovitine; Signal Transduction; Tumor Cells, Cultured

Plasminogen activator inhibitor-1 (PAI-1) is one of the target genes of hypoxia inducible factor-1alpha (HIF-1alpha). Besides being an important physiological regulator of the fibrinolytic system PAI-1 is also involved in cancer invasiveness. HIF-1alpha is expressed in various types of pulmonary cells, but the relation of PAI-1 to HIF-1alpha under hypoxic condition in these cells are not fully elucidated. We, therefore, studied the effect of hypoxia on the expression of PAI-1 in a lung cancer cell line EBC-1. The expression of HIF-1alpha protein in EBC-1 cells was enhanced by hypoxia, and this was associated with increased secretion of PAI-1. Hypoxia did not affect the levels of HIF-1alpha mRNA but enhanced the PAI-1 mRNA. Pretreatment of the cells with MG132, which inhibits the proteasomal degradation of HIF-1alpha, increased the production of PAI-1 under both normoxia and hypoxia. We conclude that hypoxia induces PAI-1 expression, in EBC-1 cells, through the stabilization of HIF-1 complex and this may be related to cancer metastasis.

Topics: Cell Hypoxia; Cysteine Proteinase Inhibitors; Dactinomycin; Gene Expression; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Immunohistochemistry; Leupeptins; Lung Neoplasms; Neoplasms, Squamous Cell; Plasminogen Activator Inhibitor 1; Transcription Factors; Tumor Cells, Cultured

Activation of signaling pathways after DNA damage induced by topoisomerase (topo) poisons can lead to cell death by apoptosis. Treatment of human nonsmall cell lung carcinoma (NSCLC-3 or NSCLC-5) cells with the topo I poison SN-38 or the topo II poison etoposide (VP-16) leads to activation of NF-kappaB before induction of apoptosis. Inhibiting the degradation of IkappaBalpha by pretreatment with the proteasome inhibitor MG-132 significantly inhibited NF-kappaB activation and apoptosis but not DNA damage induced by SN-38 or VP-16. Transfection of NSCLC-3 or NSCLC-5 cells with dominant negative mutant IkappaBalpha (mIkappaBalpha) inhibited SN-38 or VP-16 induced transcription and DNA binding activity of NF-kappaB without altering drug-induced apoptosis. Regulation of apoptosis by mitochondrial release of cytochrome c and activation of pro-caspase 9 followed by cleavage of poly(ADP-ribose) polymerase by effector caspases 3 and 7 was similar in neo and mIkappaBalpha cells treated with SN-38 or VP-16. In contrast to pretreatment with MG-132, exposure to MG-132 after SN-38 or VP-16 treatment of neo or mIkappaBalpha cells decreased cell cycle arrest in the S/G2 + M fraction and enhanced apoptosis compared with drug alone. In summary, apoptosis induced by topoisomerase poisons in NSCLC cells is not mediated by NF-kappaB but can be manipulated by proteasome inhibitors.

Topics: Apoptosis; Camptothecin; Carcinoma, Non-Small-Cell Lung; Cisplatin; DNA; DNA Damage; DNA-Binding Proteins; Enzyme Inhibitors; Etoposide; Humans; I-kappa B Proteins; Irinotecan; Leupeptins; Lung Neoplasms; NF-kappa B; NF-KappaB Inhibitor alpha; Paclitaxel; Peptide Hydrolases; Proteasome Endopeptidase Complex; Topoisomerase I Inhibitors; Topoisomerase II Inhibitors

CTLs specific for tumor antigens play a major role in immunity against cancer. Improved binding affinity of putative TAA peptides could enhance the in vivo immunogenicity of these self-altered self- tumor antigens. We examined here the efficacy of tumor vaccines composed of an altered peptide ligand of MUT-1, designated MUT-D, which exhibited significantly higher class-I allele K(b) binding affinity than its native counterpart MUT-1. The peptide was loaded on antigen presenting cells composed of the C57BL/6-syngeneic fibroblast cell line BLK.CL4. These cells were treated with proteasome inhibitor in order to shut off the degradation of proteins and the subsequent loading of endogenous peptides onto MHC class-I molecules, thus allowing for the pulsing of these cells with the modified peptide MUT-D. Proteasome-inhibited and modified peptide-loaded fibroblasts induced a peptide-specific CTL that significantly delayed primary tumor progression and protected the pre-immunized mice against the development of lung metastasis following the surgical removal of the primary tumor. Genetic modification of the fibroblasts to express the immunostimulatory cytokine IL-2 did not improve the APC function of the modified cells, nor did it result in augmentation of the potency of the vaccine. Our results suggest that the proteasome-inhibited fibroblasts pulsed with modified, high binder tumor-associated antigen peptide are good antigen-presenting cells and represent an effective form of tumor vaccine.

Topics: Animals; Antigens, Neoplasm; Cancer Vaccines; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Epitopes, T-Lymphocyte; Fibroblasts; Immunotherapy; Interleukin-2; Leupeptins; Lung Neoplasms; Male; Mice; Mice, Inbred C57BL; Multienzyme Complexes; Neoplasm Metastasis; Neoplasm Transplantation; Oligopeptides; Proteasome Endopeptidase Complex; T-Lymphocytes, Cytotoxic

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Camptothecin; Carcinoma, Non-Small-Cell Lung; DNA-Binding Proteins; Enzyme Inhibitors; Humans; I-kappa B Proteins; Irinotecan; Leupeptins; Lung Neoplasms; NF-kappa B; NF-KappaB Inhibitor alpha; Topoisomerase I Inhibitors; Transfection; Tumor Cells, Cultured

Overexpression or activation of insulin-like growth factor I receptor (IGF-IR) has been observed in many human cancers including breast, lung, colon and gastric carcinomas. We demonstrate that inhibition of the endogenous insulin-like growth factor I receptor by stable expression of a dominant-negative IGF-IR represses the transforming activity in vitro and tumorigenicity of human lung carcinoma cells A549 in vivo. The suppression of tumorigenicity in nude mice is correlated with the induction of glandular differentiation. In addition, functional inhibition of the endogenous receptor dramatically increases the sensitivity of A549 cells to a variety of apoptotic signals including UV irradiation and proteasome inhibitors. These effects are due to the formation of a stable heterocomplex of the dominant-negative receptor with the endogenous wild type receptor which reduces the kinase activity of the latter by twofold. Thus, inhibition of the IGF-IR signaling pathway not only suppresses tumorigenicity but also enhances sensitivity to apoptosis-inducing agents. Antagonizing IGF-IR signaling by promoting tumor differentiation and enhancing sensitivity to apoptotic death are potential cancer therapeutic approaches.

Topics: Acetylcysteine; Adenocarcinoma; Animals; Apoptosis; Carcinogenicity Tests; Cell Differentiation; Cell Division; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Genes, Dominant; Genes, Tumor Suppressor; Humans; Leupeptins; Lung Neoplasms; Mice; Mice, Nude; Multienzyme Complexes; Phosphorylation; Proteasome Endopeptidase Complex; Receptor, IGF Type 1; Tumor Cells, Cultured; Ultraviolet Rays

In this study we investigated the levels of two lysosomal cysteine protease proteins cathepsin B (CB) and cathepsin L (CL) and the levels of three cysteine protease inhibitor proteins stefin A (SFA), stefin B (SFB) and cystatin C (CNC) in squamous-cell lung carcinoma (SQCLC) and matched lung parenchyma specimens and examined the inhibition of CB and cathepsin C (CC) activities by endogenous inhibitors in extracts from SQCLC, lung adenocarcinoma (LAC) and lung parenchyma specimens. We found that Stage I SQCLCs contained significantly increased levels of CB protein, CB activity and SFA protein as compared to matched lungs. Neither the levels of CL protein nor the levels of SFB protein nor the levels of CNC protein in Stage I SQCLCs and the lungs were significantly different, but the levels of CB and CL proteins as well as the levels of SFA and SFB proteins showed significant positive correlation in SQCLCs. In SQCLCs as well as in the lungs the level of SFB protein was significantly higher than the level of SFA protein or the level of CNC protein. In the lungs the levels of SFA protein and CNC protein revealed a weak negative correlation trend. In extracts from SQCLCs the level of SFA protein showed a weak negative correlation with the residual CB activity (i.e. the activity remaining after extract preincubation) whereas in extracts from the lungs the level of CNC protein displayed a weak negative correlation trend with the residual CB activity and with the residual CC activity. We observed that SQCLCs and LACs contained not only a significantly increased activity of CB but also a significantly higher inhibitory potential against the activity of endogenous CB as compared to matched lungs. Leupeptin, a small inhibitor of CB, was capable to protect CB in lung carcinoma and lung parenchyma extracts from preincubation-induced inhibition, revealing an active-site directed and competitive nature of CB inhibition by endogenous cystatins. Ultrafiltration passaged protein preparations of nominal Mr < or = 30,000 obtained from extracts of SQCLCs inhibited significantly higher quantities of activity of purified bovine spleen CC than did such protein preparations from matched lungs. Reaction courses of purified bovine spleen CC that had been preincubated with such protein preparations resembled those of endogenous CC from SQCLC and lung extracts showing a slow steady-state approach. These observations and the relaxation kinetics of CC from SQCLC and lung extracts sugges

Topics: Adenocarcinoma; Adult; Aged; Animals; Carcinoma, Squamous Cell; Cathepsin B; Cathepsin C; Cathepsin L; Cathepsins; Cattle; Cystatin A; Cystatin B; Cystatin C; Cystatins; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; Endopeptidases; Female; Humans; Kinetics; Leupeptins; Lung; Lung Neoplasms; Lysosomes; Male; Middle Aged; Sensitivity and Specificity

Peptide aldehyde transition state analogue inhibitors of serine and cysteine proteases have been used to selectively inhibit proteases for which prior evidence supports a role in tumor cell metastasis. These enzymes include cathepsin B, urokinase plasminogen activator (PA), and thrombin. The inhibition constants of the peptidyl aldehyde inhibitors show that they are highly selective for a particular targeted serine or cysteine protease. The inhibitors are introduced by i.p. injection or by miniosmotic pumps into syngeneic C57BL/6 mice also given injections of B16-F10 melanoma cells, and the number of metastatic foci in the lung was determined. While the injection protocol gave an initially high but changing in vivo concentration of inhibitor over time, the minipump implant gave a constant steady state concentration of inhibitor over 5-7 days. Minipump infusion of leupeptin (acetylleucylleucylargininal), a strong inhibitor of cathepsin B at a steady state plasma concentration 1000-fold greater than its Ki(cathepsin B), gave no significant decrease in lung colonization by the B16 tumor cells. Ep475, a stoichiometric irreversible peptide inhibitor of cathepsin B-like proteases, also did not significantly inhibit metastatic foci formation. Introduction of selective inhibitors of urokinase PA, tert-butyloxycarbonylglutamylglycyl-argininal and H-glutamylglycylargininal at concentrations near its Ki, produced no significant decrease in mouse lung colonization. The selective thrombin inhibitor D-phenylalanylprolylargininal infused to a steady state concentration 100-fold greater than its Ki dramatically increased B16 melanoma colonization of mouse lung. The results indicate that neither secreted cathepsin B-like nor urokinase PA have roles in B16 colonization of mouse lung, while thrombin may have a role in preventing metastasis. These experiments do not eliminate roles for a cathepsin B-like enzyme or urokinase PA in the initial steps of the metastatic process.

Topics: Animals; Cathepsin B; Cathepsins; Cysteine Endopeptidases; Dose-Response Relationship, Drug; Endopeptidases; Female; Fibrinolysis; Leucine; Leupeptins; Lung Neoplasms; Melanoma; Mice; Mice, Inbred C57BL; Neoplasm Metastasis; Peptide Hydrolases; Plasminogen Activators; Plasminogen Inactivators; Protease Inhibitors; Serine Endopeptidases; Thrombin; Thrombosis

The inhibitory effects of protease inhibitors on blood-borne metastasis in male Donryu rat lung were studied. Injection i.v. of 10(6) Yoshida ascites hepatoma AH7974 cells induced about 118 +/- 92 (S.D.) metastatic foci in rat lung after 3 weeks. Leupeptin (50 mg/kg body weight twice a day), injected i.p. from 2 days before to 4 days after the inoculation of tumor cells, reduced the number of metastatic foci to about 49 +/- 45 (p less than 0.005). Leupeptin also suppressed the formation of metastatic foci of Yoshida ascites hepatoma AH100B cells (p less than 0.001). Elastatinal (100 mg/kg body weight twice a day) and chymostatin (100 mg/kg body weight once a day) did not inhibit formation of metastatic foci of AH7974 cells. Injection i.v. of 10(6) AH7974 cells induced pulmonary thrombi within 1 hr. Leupeptin (50 mg/kg body weight twice a day) reduced the number of thrombi from 1298 +/- 395 to 646 +/- 218, when injected i.p. for 2 days before the inoculation of the cells (p less than 0.005). Chymostatin and elastatinal did not significantly change the number of pulmonary thrombi. These results indicate that leupeptin inhibited metastasis formation and suggest that this effect may be due to the inhibition of thrombus formation after the arrest of circulating tumor cells.

Topics: Animals; Leupeptins; Liver Neoplasms, Experimental; Lung Neoplasms; Male; Neoplasm Metastasis; Neoplasm Transplantation; Neoplastic Cells, Circulating; Protease Inhibitors; Pulmonary Embolism; Rats

Topics: Animals; Antineoplastic Agents; Aprotinin; Enzyme Inhibitors; Lactones; Leupeptins; Lung Neoplasms; Lysosomes; Male; Mice; Mice, Inbred C57BL; Neoplasm Metastasis; Neoplasms, Experimental; Pepstatins